Cystatin-related epididymal spermatogenic protein (CRES, also CST8) is expressed in both the testis and epididymis and found associated with spermatozoa. It appears as nonglycosylated (14 and 12 kDa) and glycosylated isoforms (19 and 17 kDa). The role of CRES remains enigmatic and is dependent on localization of its isoforms, which is the objective of this study. Our initial approach was to investigate testicular origins of these isoforms by immunohistochemistry and immunogold electron microscopy. We confirmed previous reports that CRES was expressed in the cytoplasm of elongating spermatids from step 8 to step 16. However, we noticed that the pattern of testicular expression was reminiscent of outer dense fiber (ODF) and fibrous sheath (FS) proteins. Western blot analysis of extracts of cauda epididymal sperm revealed a nonionic, detergent-insoluble 14-kDa CRES isoform. To further pinpoint and confirm CRES localization we separated sperm heads and tails and extracted the tails with progressively harsher protein solubilizing agents. Western blots of these sequential extracts, designed to progressively remove the mitochondrial sheath and the ODFs or FS, detected a CRES-immunoreactive 14-kDa band associated with the accessory fibers of the tail. Immunogold labeling was concentrated over growing ODFs in the testes and persisted in spermatozoa. This study discovers a CRES isoform that assembles as part of the ODFs during the elongation and maturation phases of spermiogenesis and is retained as a covalently bound component of the ODFs in spermatozoa.